Environmental agents such as ultraviolet light, ionizing radiation, air pollution, chemotherapeutic drugs, and chemicals found in cigarette smoke, combined with internal factors produced during processes of normal cellular metabolism generate reactive oxygen species (ROS) in cells. ROS cause damage to cellular DNA, which if not properly repaired, can trigger genome instability and the progression of neurodegenerative disorders, aging, and cancer. This proposal seeks to study the removal of ROS-generated DNA damage via the base excision repair (BER) pathway in mitochondrial DNA (mtDNA), which is more susceptible than its nuclear counterpart to oxidative stress owing to its proximity to sites of ROS generation. DNA glycosylases play a critical role in initializing BER by excising damaged base and mediating other aspects of the repair process via essential protein:protein interactions. The mentored K99 phase of the proposal seeks to delineate a role for two Nei-like (NEIL) DNA glycosylases, NEIL 1 and NEIL 2, in the repair of mtDNA. Aim 1 will focus on understanding effects on mtDNA damage in the absence of expression of the NEIL enzymes using mouse embryo fibroblasts. Experiments involving the determination of mitochondrial function in real-time using Seahorse technology and qPCR techniques to measure the extent of DNA damage will be performed in the Van Houten laboratory (University of Pittsburgh). The second aim, also initiated during the mentored phase, will focus on the study of critical protein:protein interactios between the NEIL enzymes and mitochondrial proteins involved in mitochondrial genome maintenance. This work will be performed using a combination of co-immunoprecipitation, yeast two- hybrid analysis, and purified proteins to validate the results obtained. The R00 independent phase will consist of structure-function studies to investigate the molecular basis of protein:protein interactions mediated by the NEIL enzymes. A multi-disciplinary approach using small-angle X-ray scattering to characterize the complexes formed between the NEIL enzymes and mitochondrial proteins and X-ray crystallography to determine the crystal structures of the complexes will be undertaken. Mutational analysis will be used to test the functional relevance of these interactions. The proposed research supports the mission of the NIEHS by studying the effects of environmental and other agents on the progression of diseases associated with mtDNA damage. The long-term goal of the proposed research is to design ways to hinder key protein:protein interactions, which could inhibit mitochondrial BER and prevent aberrant cell growth.